Sustained release

ABSTRACT

Disclosed are dispensing systems for releasing a substance at a substantially constant rate and methods for its manufacture. The composition comprises semipermeable capsules containing the material to be released. The capsules comprise membranes having pores of dimensions sufficient to control the kinetics of release provided there is maintained a large intracapsular concentration of the substance relative to the concentration desired in the extracapsular environment. The compositions may be produced by forming permeable capsules, suspending the capsules in a solution containing a high concentration of the substance to load the intracapsular volume, and then post-treating the capsules to reduce the size of the pores.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of Ser. No. 485,471 filed Apr. 15,1983, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to microencapsulation. More particularly, itrelates to a method of loading permeable microcapsules and a compositionof matter comprising a permeable microcapsule containing one or moresubstances which may be released from the intracapsular volume at asubstantially constant rate for a significant time.

Compositions of matter capable of sustained release of drugs,fertilizers, and the like are well known in the art. Generally, suchcompositions comprise a solid carrier in which the substance to bereleased is adsorbed or trapped as plural separate phases. When suchcompositions are placed in the environment in which they are intendedfor use, the outer layers of the solid carrier dissolve releasing aportion of the substance of interest. If the solid carrier dissolvesslowly, then the substance of interest is released slowly or in discretebursts over time. In use, the rate of release of the substance from suchcompositions necessarily is dependent upon the solubility of the carrierparticle material, on the surface area of the carrier particle that atany given time is exposed to the environment, and often on thesolubility of the substance in the environment of use. These factorstypically result in a release rate which decreases over time.

In situations where it is desired, e.g., to release a drug at a constantrate into the circulatory system, the foregoing approach based onsolubility properties cannot be used. In such situations, implantablemechanical perfusion pumps and similar devices have been suggested.

SUMMARY OF THE INVENTION

It has now been discovered that sustained release at a substantiallyconstant rate can be achieved for essentially any substance by providinga solid or liquid reservoir of the substance within the interior volumeof one or more capsules. Each of the capsules comprise a membrane havingpores of dimensions sufficient to limit the rate at which molecules ofthe substance pass therethrough. The capsules are used in an environmentwhich depletes the substance, e.g., by chemical modification, sorption,metabolic breakdown, ingestion, diffusion, or simple removal by fluidflow.

The concept of the invention involves loading permeable capsules with areservoir of the substance of interest at a concentration sufficient toprovide an osmotic pressure above a threshold level, and controlling thepore size of the capsule membranes so that passage of molecules of thesubstance through the membrane becomes the rate-limiting factor indispensing the substance into the extracapsular environment. In oneimportant embodiment, the substance of interest is a solid containedwithin the capsule together with a solvent for the solid. Once the solidand its solvent reach equilibrium, a reservoir of molecules of thesolvent is available to provide a continuous, substantially constantmigration of molecules through the membrane. In another embodiment, thesubstance is contained within the capsule as a solution at aconcentration in excess of the desired extracapsular concentration.Molecules of the substance are released into the extracapsularenvironment at a substantially constant rate until the intracapsularconcentration drops to a level where the intracapsular osmotic pressureis insufficient to support the membrane dependent transfer rate.

The intracapsular concentration of the substance to be dispensed shouldbe quite high, typically at least two orders of magnitude greater thanthe desired extracapsular concentration, and more preferably at leastthree. Generally, the higher the intracapsular concentration the longerthe constant release rate can be sustained. If the capsules are placedin an enviornment with no mechanism for removing the substance, theneventually the intracapsular and extracapsular concentration willequalize. Thus, capsules may be stored as a suspension in a volume of acompatible solvent containing a concentration of the substancesubstantially equal to or greater than the intracapsular concentration.In this circumstance net flow of the substance out of the capsules isprevented.

A preferred process for producing the dispensing systems of theinvention involves forming capsules having membranes which permittraverse of the substance to be loaded therewithin, suspending thecapsules in a concentrated solution of the substance one or more timesto diffuse the substance into the intracapsular volume, and thereafterfurther treating the capsule to reduce the dimensions of the pores inthe membrane.

A preferred method of making the capsules involves formingshape-retaining spheres, e.g., on the order of 0.1 mm to 3 mm indiameter from a water-soluble polymer containing plural anionic orcationic groups and cross-linking surface layers of the by contact witha polymer having plural groups of a charge opposite that of thewater-soluble polymer. After loading, the capsule is again treated withthe same or a different cross-linking polymer to reduce the dimensionsof the pores.

One outstanding advantage of the process and dispensing system of theinvention is that it can be modified readily to dispense essentially anysubstance desired at a controlled, substantially constant rate. Anotheradvantage is that the capsule membranes can be fabricated from non-toxicmaterials that produce no significant immunological response, andaccordingly are well suited for administration of biologically activematerials such as hormones, antibodies, antigens, enzymes, lymphokines,vaccines, and natural or synthetic drugs via implantation in an animalbody. Additional uses for the dispensing system of the invention includethe sustained release of fertilizers, pesticides, fungicides, planthormones and growth factors, flavors, perfumes, preservatives, andnutrients such as tissue culture nutrients.

In another aspect, the invention comprises a method of administering asubstance to an animal comprising implanting, preferably by injection,one or more capsules of the type described containing the substance.

Accordingly, it is an object of the invention to provide a novelcomposition of matter capable of sustained release of a substance intoan environment at a substantially constant rate. Another object is toprovide a method of loading microcapsules. Another object is to releasea biologically active material into an animal body at a substantiallyconstant rate. Yet another object is to provide an improved method ofcontrolling the pore size of a microcapsule membrane.

These and other objects and features of the invention will be apparentfrom the description which follows and from the drawing.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph of measured extracapsular insulin concentration versustime illustrating the substantially constant release rate of insulinusing a composition embodying the invention which employs crystallineinsulin;

FIG. 2 is a graph of the cumulative extracapsular insulin concentrationachieved using the preparation of FIG. 1.

FIG. 3 is a graph of cumulative insulin concentration in anextracapsular medium versus time using a composition of the inventionemploying a solution of insulin;

FIG. 4 is a graph of myoglobin concentration versus time illustratingthe substantially constant rate of release of myoglobin; and

FIG. 5 is a graph of hemoglobin concentration versus time.

DESCRIPTION

The system of the invention involves the production of spheroidalcapsules each of which comprise membranes having pores of dimensionssmall enough to hinder passage of molecules from a reservoir of asubstance within the capsule. The rate at which molecules are releasedinto the extracapsular volume is controlled by the kinetics of theirpassage through the membrane.

Well established principle of mass transfer across a porous membranedictate that where the pores of the membrane are sufficiently large, andthere is a concentration gradient across the membrane, solute will flowfrom the more concentrated region to the less concentrated region at asteadily falling rate until both sides of the membrane are inequilibrium. This equilibrium will also be attained where the pores ofthe membrane are of dimensions sufficient to hinder passage of massacross the membrane, provided some finite mass transfer occurs. However,in the latter situation, where the interior of a capsule membrane has ahigh concentration of the solute and the outside is at a lowconcentration, the passage across the membrane may be the diffusion ratelimiting step. Accordingly, until such time that the concentrationoutside the capsule is high enough to produce a significant balancingosmotic pressure, or such time that the concentration of solute withinthe capsule becomes insufficient to generated sufficient osmoticpressure to drive the steady diffusion across the membrane, the rate atwhich solute molecules diffuse out of the capsule will be substantiallyconstant.

The dispensing system of the invention is designed for use where somephysical, chemical, or biological mechanism depletes the soluteconcentration immediately outside the capsule membranes. Accordingly,there is no significant buildup of the extracapsular concentration.Thus, for as long as there is a sufficient concentration of solutewithin the capsule, the diffusion kinetics are controlled by the capsulemembrane. Once an equilibrium outflow of the solute is attained,diffusion proceeds at a substantially constant rate until theintracapsular concentration falls below a level where it influences thediffusion kinetics.

From the foregoing it is apparent that the ability to fabricate capsulemembranes having the desired permeability characteristics, and theability to load the capsules with a high concentration of the substanceto be released are both important to the practice of the invention.

Procedures for encapsulation which enable some control of membraneporosity are known in the art, see, for example, U.S. Pat. No.4,352,883. It has been discovered that by suitably modifying theprocedures disclosed in the foregoing U.S. patent, the disclosure ofwhich is incorporated herein by reference, it is possible to determineempirically a procedure for producing capsule membranes with therequired permeability characteristics. The principles used to controlmembrane porosity are disclosed herein. Furthermore, several proceduresfor loading the capsules with a high concentration of the substance tobe released have also been developed.

In the encapsulation technique of the '883 patent, a water-solublepolycationic or polyanionic polymer is formed into a spheroidal,shape-retaining droplet which suspends the material to be encapsulated.Thereafter, the droplet is exposed to a polymer having the oppositecharge to cross-link surface layers of the droplet by the formation ofsalt bridges between the polyanionic and polycationic polymers. Thisprocedure produces a porous membrane.

The material to be encapsulated is prepared in accordance withwell-known techniques in finely divided or solution form, and awater-soluble polyanionic or polycationic material which can bereversibly gelled is added to the suspension or solution in lowconcentration. The droplet containing the material to be encapsulated isimmediately rendered water-insoluble and gelled, at least in surfacelayers. Thereafter, the shape-retaining capsules are provided with apermanent semipermeable membrane. Where the material used to form thetemporary capsules permits, the capsule interior may be reliquifiedafter formation of the permanent membrane. This is done byreestablishing the conditions in the medium at which the material issoluble.

The material used to form the gelled droplets may be any water-solublematerial which, by a change in the surrounding temperature, pH, or ionicenvironment or concentration, can be converted to a shape-retainingmass. The material must also comprise, for purposes of this invention,plural, easily ionized groups, e.g., carboxyl or amino groups which canreact by salt formation with polymers containing plural groups whichionize to form species of the opposite charge. Preferred materials forforming the temporary capsule are water-soluble, natural or syntheticpolysaccharides. Sodium alginate is most preferred. Other usablepolysaccharides include carboxylated fractions of guar gum, gum arabic,charageenan, pectin, tragacanth gum, or xanthan gum.

These materials comprise glycoside-linked saccharide chains. Manycontain free acid groups, which are often present in the alkali metalion form, e.g., sodium form. If a multivalent ion such as calcium orstrontium is exchanged for the alkali metal ion, the liquid,water-soluble polysaccharide molecules are "cross-linked" to form awater-insoluble, shape-retaining gel which can be resolubilized onremoval of the ions by ion exchange or via a sequestering agent. Whileessentially any multivalent ion which can form a salt with thesaccharides is operable, physiologically compatible ions, e.g., calcium,is preferred for purposes of making capsules designed for the sustainedrelease of drugs. Other polysaccharides can be switched between thewater-soluble and gelled, water-insoluble state simply by changing thepH of the medium in which they are dissolved.

In the next step of the process, a semipermeable membrane is depositedabout the surface of the gelled droplets. The preferred technique forforming the membrane is to cross-link surface layers of the droplets bysubjecting them to an aqueous solution of a polymer containing groupsreactive with functionalities in the gel molecules. Certain long chainquaternary ammonium salts may be used for this purpose in somecircumstances. When acidic gels are used, polymers containing acidreactive groups such as polyethylenimine, polyvinyl amine, andpolypeptides such as polylysine may be used. In this situation, thepolysaccharides are cross-linked by interaction between the carboxylgroups and the amine groups.

It has been discovered that permeability can be controlled sufficientlyfor purposes of the invention by selecting the molecular weight of thecross-linking polymer used, adjusting the concentration of thecross-linking polymer, and by cross-linking in two or more separatestages using the same or a different cross-linking agent. Generally, asolution of polymer having a low molecular weight, in a given timeperiod, will penetrate further into the temporary capsules than will ahigh molecular weight polymer. In general, the higher the molecularweight and the less penetration, the larger the pore size. Broadly,polymers within the molecular weight range of 3,000 to 100,000 daltonsor greater may be used, depending on the duration of the reaction, theconcentration of the polymer solution, and the degree of permeabilitydesired. The currently preferred molecular weight range is 20,000 to80,000. Post-treating the capsule with the same or a differentcross-linking polymer has the effect of reducing the dimensions of thepores and thickening the membrane.

At this point in the encapsulation, capsules may be collected whichcomprise a permanent semipermeable membrane surrounding a gelledsolution of polysaccharide containing the core material. If masstransfer is to be promoted within the capsules and across the membranes,it is preferred to reliquify the gel to its water-soluble form. If lowmolecular weight polysaccharide is used, much of the excess material canbe removed from within the membrane by diffusion. The liquification maybe conducted by reestablishing the conditions under which thewater-soluble polymer is a liquid, e.g., changing the pH of the mediumor removing the calcium or other multifunctional cations. In the gelswhich are insoluble in the presence of multivalent cations, the mediumin the capsule can be resolubilized simply by immersing the capsules inphosphate buffered saline, which contains alkali metal ions and hydrogenions. Monovalent ions exchange with the calcium or other multifunctionalions when the capsules are immersed in the solution with stirring. Othersalts, e.g., sodium citrate, may be used for the same purpose.

Lastly, depending on the type of semipermeable membrane formationtechnique employed, it is often desirable to treat the capsules so as totie up free amino groups or the like which would otherwise impart to thecapsules a tendency to clump. This can be done, for example, byimmersing the capsules in a dilute solution of sodium alginate.

In this encapsulation technique it is difficult to synthesize themembranes while simultaneously retaining a high concentration of thesubstance to be encapsulated within the forming membrane unless thesubstance has a high molecular weight i.e., ≧300,000. One solution tothis problem is to encapsulate a solid material. After the capsules arecompleted, they are placed in the environment of use where solventdissolves the intracapsular solid. Thus, there is produced within theintracapsular volume a reservoir of solute, at a concentration relatedto the solubility product of the solid in the solvent. The reservoir ismaintained until the solid dissolves.

Another loading technique involves forming permeable capsules which haveno initial loading or relatively low loading and thereafter suspendingthe capsules in a concentrated solution of the substance to beencapsulated. Thus, the capsules are loaded from the outside.Thereafter, the capsules are again treated with a polymericcross-linking agent of the type described above to reduce further thedimensions of the pores.

When employing this outside loading technique, it is preferred that thesolution in which the capsules are suspended be as concentrated aspossible; at least three orders of magnitude greater than theextracapsular concentration desired during use of the compositions, andpreferably even higher. Especially where the substance to be loaded hasa high viscosity or a low solubility, it is preferred to repeat theoutside loading procedure one or more times. It is also preferred toproduce the capsule membrane at the outset so as to provide pores ofdimensions sufficient to retard the passage of the solute into thecapsule. This lengthens the loading time, but also minimizes leakage ofthe loaded materials during the time the second cross-linking step isbeing conducted to further reduce the pore dimensions and thicken themembrane.

From the foregoing it should be apparent that some experimentation willbe required in the design of any specific composition for dispensing agiven substance. However, in view of this disclosure those skilled inthe art will be able to produce a variety of specific compositionshaving a desired constant rate of release. In some cases it is difficultto set the constant release rate of a given microcapsule at a specificdesired level. However, it is relatively simple to control the averagerate of release of a large number of capsules at an arbitrary value.Thus, dosage can be controlled by supplying a number of capsules whichtogether release the desired quantity of the substance to be dispensedat a constant rate over a significant time.

Control of the permeability characteristics of the membrane is exercisedby varying (1) the materials used to make the membranes, (2) the polymerconcentration of the cross-linking solution, (3) the molecular weight ofthe cross-linking polymer or polymers, (4) the duration of thecross-linking step, and (5) the number of cross-linking steps conducted.While the effect on permeability characteristics of any one variableisolated from the others is difficult to define precisely, the followinggeneral comments can be made. Materials which have a high density ofcharged groups tend to result in highly cross-linked membranescharacterized by smaller pore dimensions. Higher concentrations ofpolymer and longer exposures to the polymer tend to reduce thedimensions of the pores in the dimensions of the plane of the membrane,and produce thicker membranes with greater pore lengths. Low molecularweight materials tend to penetrate further into the gel. This typicallyresults in reducing the permeability of the membrane. Cross-linkershaving a molecular weight below about 3,000 daltons tend to permeate andreact with substantially the entrire gel sphere. Higher molecular weightmaterials form a relatively thin membrane on surface layers of the gel.Residual polysaccharide is available to bond with additional quantitiesof the cross-linker in multiple step cross-linking processes. The use ofsuch multiple separate cross-linking steps tends to reduce the poresize.

The membrane is believed to comprise a matrix of polymers cross-linkedwith ionic bonds. The polymers define random intermolecular spaces whichcommunicate with each other to form tortuous path pores through themembrane. It is also believed that both the pore dimensions and theeffective length of the pore across the membrane influence the kineticsof molecular diffusion. Molecules in the volume of the pores presumablyundergo many random collisions which in the aggragate determine theaverage time it takes for a molecule of a given effective dimension totraverse the membrane.

The substances which may be encapsulated in accordance with theinvention to produce compositions characterized by substantiallyconstant rate, sustained release can vary widely. The only limitingfactors appear to be that it is difficult to produce a membrane thatwill be the dominating factor in controlling the diffusion rate in thecase of very low molecular weight materials, e.g., 200 daltons or below.Also, capsule membranes uniformly permeable to substances having amolecular weight greater than about 10⁶ daltons are difficult tosynthesize.

The invention will be further understood from the following non-limitingexamples, wherein all percentages are given in a weight/volume basis(g/ml).

EXAMPLE 1 Crystalline Insulin Capsules

Gelled spheres were formed from 1.2% sodium alginate (NaG) in 0.9% NaClcontaining 20 mg/ml (500 units) crystalline insulin. The NaG-insulinsuspension was well mixed and the gell spheres prepared immediately sothat the insulin was uniformly distributed in the NaG. Liquid spheroidaldrops of the suspension were then immersed in a 1.5% CaCl₂ /H₂ O gellingsolution. After formation, the gelled spheres were allowed to remain inthe CaCl₂ colution for three minutes and then treated as describedbelow. All treatment and wash solutions had a volume of ten times thevolume of the original NaG solution.

1. Treat with 0.6% CaCl₂ in 0.45% NaCl for five minutes, maintaining thegelled spheres in suspension.

2. Treat with 0.3% CaCl₂ in 0.68% NaCl for five minutes, maintaining thegelled spheres in suspension.

3. Wash twice with saline.

4. Treat with 60,000 molecular weight poly-1-lysine (PLL) in saline (13mg PLL hydrobromide/dl) for five minutes, maintaining the capsules insuspension to form a membrane.

5. Wash twice in saline.

6. Wash with CHES buffer (2% 2(N-cyclohexylamino) ethanesulfonic acid in0.6% NaCl, pH 8.2) diluted 1:20 with 1.1% CaCl₂.

7. Wash twice with saline.

8. Treat with 0.11% polyvinylamine (PVA, Polysciences, molecular weightrange 50,000-150,000) in saline, pH 7.0, for five minutes, maintainingthe capsules in suspension.

9. Wash three times in saline.

10. Treat with 0.6% NaG for five minutes, maintaining the capsules insuspension.

11. Wash twice in saline.

12. Treat with 0.5% PVA in saline for five minutes maintaining thecapsules in suspension.

13. Wash three times in saline.

14. Store capsules in saline or 2.5% albumin in saline to stabilize theinsulin.

The capsules were suspended in saline and the extracapsularconcentration of insulin was assayed daily. As shown in FIG. 1,extracapsular concentration per day ranged uniformly between about 3 and4 units of insulin/ml of extracapsular saline. As shown in FIG. 2, thecumulative insulin concentration in the extracapsular medium rises at asubstantially constant rate.

EXAMPLE 2 Liquid Insulin Capsules

Blank gel spheres were made by dropping liquid spheres comprising 1.2%NaG in 0.9% saline into a 2.0% CaCl₁₂ /H₂ O gelling solution. The CaCl₂solution has a volume of 25 times that of the NaG solution. Afterformation, the gelled spheres were left in the CaCl₂ for three minutesthen treated as described below. All treatments and wash solutions had avolume of ten times the volume of the original NaG solution.

1. Wash with CHES buffer diluted 1:20 with 1.1% CaCl₂.

2. Wash with 1.1% CaCl₂.

3. Treat with 60,000 molecular weight poly-1-lysine (PLL) in saline (6.7mg PLL hydrobromide/dl) for four minutes, maintaining the capsules insuspension.

4. Wash with the CHES buffer-CaCl₂ solution as in step 1.

5. Treat with 0.11% polyvinylamine (PVA) in saline, pH 7.0,for fourminutes, maintaining the capsules in suspension.

6. Wash three times with saline.

7. Treat with 0.6% NaG in saline for four minutes, maintaining thecapsules in suspension.

8. Wash three times in saline.

This completes the formation of the blank microcapsules. These capsulesmay be stored in saline until ready for loading and further treatment ofthe membranes.

The capsules were loaded with insulin by suspending them in an equalvolume of 500 units insulin/ml for 15 to 18 hours at 37° C. As much aspossible of the extracapsular loading solution was then removed and thecapsules were quickly washed once in four times their volume of saline.Immediately after washing the capsules were treated further using theprocedure described below. All wash and treatment volumes were fourtimes the volume of the capsules.

1. Treat the capsules with 0.33% PVA in saline, pH 7.0, for six minuteskeeping the capsules in suspension.

2. Wash three times in saline.

3. Treat with 0.06% NaG in saline for four minutes.

4. Wash and store the capsules in saline.

The finished capsules were suspended in saline containing 2.5% albumin.Extracapsular insulin concentration was measured daily. The results areillustrated in FIG. 3. As shown, the cumulative insulin concentrationincreased at a substantially constant rate for a 5 day period.

EXAMPLE 3 Myoglobin Capsules

Blank gel spheres were made by dropwise addition of a 1.2% sodiumalginate solution in saline, to a 1.5% CaCl₂ /H₂ O gelling solution. TheCaCl₂ solution had a volume of 25 times the volume of the NaG solution.After formation, the spheres were left in the CaCl₂ solution for fourminutes and then treated as described below. All the solutions had avolume of ten times the volume of the original NaG solution.

1. Transfer to 0.6% CaCl₂ in 0.45% NaCl and allow to remain for fiveminutes.

2. Transfer to 0.3% CaCl₂ in 0.68% NaCl and allow to remain for fiveminutes.

3. Wash once in 0.9% saline.

4. Treat with 60,000 molecular weight poly-1-lysine (PLL) in saline(13.3 mg PLL hydrobromide/dl) for five minutes, maintaining the capsulesin suspension.

5. Wash with saline three times.

6. Wash with CHES buffer diluted 1:20 with 1.1% CaCl₂ /H₂ O.

7. Treat with 0.17 polyvinylamine (PVA) in saline, pH 7.0, for fiveminutes, maintaining the capsules in suspension.

8. Wash three times with saline.

9. Treat with 0.06% NaG in saline for four minutes maintaining thecapsules in suspension.

10. Wash two times in saline.

11. Treat with citrate solution (0.75% sodium citrate pH 7.4 in water).

12. Wash in 0.23% NaCl.

The blank capsules produced as disclosed above were loaded withmyoglobin by suspending equal volumes of capsules and 2,000 mg/dl ofmyoglobin in saline for 15 to 18 hours. The capsules were then washedwith saline until the supernatant was clear, and then further treatedusing the procedure described below. All treatment and wash volumes werefour times the volume of the capsules.

1. Treat with 0.5% PVA in saline for five minutes maintaining thecapsules in suspension.

2. Wash three times in saline.

3. Treat with 0.6% NaG in saline for four minutes maintaining thecapsules in suspension.

4. Wash and store the capsules in 0.23% NaCl.

The capsules were then suspended in saline and the extracapsularsolution was periodically assayed for myoglobin content. The results areshown in FIG. 4. As illustrated, the cumulative myoglobin concentrationincreased at a substantially constant rate for nine days. These capsulesreleased approximately 5-6 mg myoglobin per dl per day.

EXAMPLE 4 Hemoglobin Loaded Capsules

Blank gelled spheres were made by dropwise addition of a 0.9% sodiumalginate (Sigma) solution in water to a 2% CaCl₂ aqueous gellingsolution. The CaCl₂ solution had a volume of 25 times the volume of theNaG solution. After formation, the spheres were left in the CaCl₂solution for five minutes and then treated as described below. Alltreatment and wash solutions had a volume of ten times the volume of theoriginal NaG solution.

1. Wash with CHES buffer diluted with 1:20 with 1.1% CaCl₂.

2. Treat with 40,000 molecular weight poly-1-lysine in 0.9% NaCl (6.7 mgPLL hydrobromide/dl) for four minutes, maintaining the capsules insuspension.

3. Wash with the CHES buffer CaCl₂ as in step 1.

4. Wash three times in 0.9% saline.

5. Treat with 0.11% polyvinylamine (PVA) in saline pH 7.0 for fourminutes, maintaining the capsules in suspension.

6. Wash three times in saline.

7. Treat with 0.06% NaG in saline for four minutes, maintaining thecapsules in suspension.

8. Wash with saline three times.

9. Treat with 0.75% trisodium citrate in 0.7% NaCl pH 7.4 for sixminutes.

10. Wash three times with saline.

The finished capsules are stored in saline until ready for loading andsealing.

The capsules were loaded with hemoglobin by suspending them in an equalvolume of at least a 10% hemoglobin solution for 15 to 18 hours. Toincrease the internal hemoglobin concentration even higher, thehemoglobin solution was replaced and the above loading step wasrepeated. After loading, as much as possible of the loading solution wasremoved and the capsules were washed quickly in saline until thesupernate was nearly clear (a small amount of hemoglobin will slowlyleak out of the capsules at this stage). Immediately after this wash,the capsules are further treated using the procedures described below.All wash and treatment volumes were four times the volume of thecapsules.

1. Treat the capsules with 0.17% PVA in saline, pH 7.0, for threeminutes, maintaining the capsules in suspension.

2. Wash three times in saline.

3. Treat with 0.03% NaG in saline for four minutes.

4. Wash three times and store the capsules in saline.

The hemoglobin capsules were suspended in saline and the extracapsularhemoglobin, concentration was assayed periodically. The results are setforth in FIG. 5. As shown, the capsules released approximately equaldaily quantities of hemoglobin and over a 14 day period.

The foregoing exemplary systems demonstrate that in accordance with theprocesses disclosed herein it is possible to produce compositions ofmatter which release at a substantially constant rate substances in thelow, medium, and moderately high molecular weight range. Insulin has amolecular weight on the order of 6×10³, myoglobin has a molecular weighton the order of 1.8×10⁴, and hemoglobin has a molecular weight on theorder of 7×10⁴.

Since, as disclosed above, the capsule membranes can be made withphysiologically compatible, non-toxic materials such as polypeptides andpolysaccharides, the capsules produce no significant detectableimmunlogical response when injected into, e.g., a body cavity, under theskin, or into muscle tissue of an animal. Accordingly, variousbiologically active materials may be administered to animals at asubstantially constant rate by implanting, preferably by injection, oneor more capsules of the type described above. A substantially constantrate of release can be achieved for a significant period of timeprovided that the extracapsular concentration of the substanceimmediately adjacent the capsules does not build up to a levelsufficient to alter the kinetics of release.

The compositions can be stored by suspending the capsules in a minimumvolume of a solution of the substance contained in the intracapsularvolume. Water-containing capsules may be suspended in a lipophilicvehicle to achieve the same purpose.

The invention may be embodied in other specific forms without departingfrom the spirit and scope thereof.

What is claimed is:
 1. An injectable dispensing system for releasing asubstance at a substantially constant rate in an environment immediatelyadjacent said dispensing system that depletes said substance, saidsystem comprising:a microcapsule having a membrane comprising a matrixof polycationic polymer salt bonded to a polyanionic polymer whichdefines an intracapsular volume, said membrane having a multiplicity ofpores of dimensions sufficient to limit the rate at which the moleculesof said substance pass therethrough from said intracapsular volume; anddisposed within said volume, for purposes of maintaining a givenextracapsular concentration in said environment, a concentration of aidsubstance at least two orders of magnitude greater than said givenextracapsular concentration which results in an osmotic pressuresufficient to sustain said limited rate when said capsule is placed insaid environment.
 2. The system of claim 1 wherein said substance is asolid disposed within said intracapsular volume in a solvent for saidsolid.
 3. The system of claim 2 wherein said solid is sparingly solublein said solvent.
 4. The system of claim 1 wherein said substancecomprises a biologically active material.
 5. The system of claim 4wherein said biologically active material is selected from the groupconsisting of hormones, enzymes, antibodies, vaccines, drugs, andlymphokines.
 6. The system of claim 1 further comprising, for storagepurposes, means defining an extracapsular volume of said substanceadjacent the exterior of said capsule, said extracapsular volumecontaining a concentration of said substance sufficient to substantiallyprohibit a net flow of said substance from said intracapsular volume tosaid extracapsular volume.
 7. The system of claim 1 wherein saidmembrane defines a sphere having a diameter between about 0.1 and 3.0mm.
 8. A process for administering a substance to an animal at asubstantially constant rate for purposes of maintaining a givenextracapsular concentration of said substance adjacent the exterior ofsaid capsule within said animal, said process comprising the steps of:A.providing a dispensing system comprising: a microcapsule having amembrane comprising a matrix of polycationic polymer salt bonded to apolyanionic polymer which is physiologically compatible with said animaland which defines an intracapsular volume, said membrane having amultiplicity of pores of dimensions sufficient to limit the rate atwhich the molecules of said substance pass therethrough from saidintracapsular volume; and disposed with said volume, a concentration ofsaid substance at least two orders of magnitude greater than said givenextracapsular concentration which results in an osmotic pressuresufficient to sustain said limited rate when said capsule is placed insaid animal; and B. Injecting said dispensing system within the body ofsaid animal.
 9. The process of claim 8 wherein said dispensing systemcomprises a plurality of said capsules.
 10. The process of claim 8wherein said substance is a solid disposed within said intracapsularvolume in a solvent for said solid.
 11. The process of claim 10 whereinsaid solid is sparingly soluble in said solvent.
 12. The process ofclaim 8 wherein said substance comprises a biologically active material.13. The process of claim 12 wherein said biologically active material isselected from the group consisting of hormones, enzymes, antibodies,vaccines, drugs, and lymphokines.
 14. A dispensing system for releasinga substance at a substantially constant rate in an environmentimmediately adjacent said dispensing system that depletes saidsubstance, said substance comprising:a microcapsule having a membranecomprising a matrix of polycationic polymer salt bonded to a polyanionicpolymer which defines an intracapsular volume, said membrane having amulitplicity of pores of dimensions sufficient to limit the rate atwhich the molecules of said substance pass therethrough from saidintracapsular volume; and disposed within said volume, for purposes ofmaintaining a given extracapsular concentration in said environment, aconcentration of said substance at least two orders of magnitude greaterthan said given extracapsular concentration which results in an osmoticpressure sufficient to sustain said limited rate when said capsule isplaced in said environment.
 15. The system of claim 14 wherein saidsubstance is selected from the group consisting of plant fertilizers,pesticides, fungicides, and plant hormones.
 16. The system of claim 14wherein said substance is selected from the group consisting of flavors,perfumes, and preservatives.
 17. The system of claim 14 wherein saidsubstance is a tissue culture nutrient.